Increased surface expression of CD18 and CD11b in leukocytes after tourniquet ischemia during elective hand surgery

The surface expression of β2-integrins was investigated in leukocytes from patients undergoing ischemia induced by tourniquet application for elective hand surgery. Blood samples were obtained before initiation, at the end of ischemia, and after 15 minutes of reperfusion from ischemic and contralateral arms of five patients. Comparable expression of CD18, CD11a, CD11b, and CD11c could be detected by immunofluorescence in leukocytes from samples drawn from either arm before tourniquet application. In contrast, a significant increase in the expression of CD18 was detectable in monocytes, granulocytes, and lymphocytes from the ischemic arm compared with that in the nonischemic contralateral control, at the end of the ischemia time (80 ± 16 minutes). A significantly increased expression of CD11b, but not CD11a or CD11c, determinants was also observed in granulocytes and monocytes. Concomitantly, a significant reduction in the percentages of granulocytes in samples from ischemic areas was detectable. After 15 minutes of reperfusion, differences in the expression of these adhesion molecules were no longer significant. The expression of the genes encoding interleukins IL-1α, IL-1β, and IL-6 and tumor necrosis factor alpha (TNFα) proinflammatory cytokines was also studied by reverse polymerase chain reaction (rPCR) in peripheral blood mononuclear cells (PBMCs) obtained from the same samples in three patients. IL-1β or IL-6 gene expression was never observed. Expression of IL-1α and TNFα genes, as detected in two patients, was not related with induction of ischemia. However, in these patients expression of one or both these genes was observed in samples derived from the ischemic but not the control arm after 15 minutes of reperfusion. These data document that overexpression of adhesion molecules and sequestration of leukocytes take place following short ischemia times, as routinely applied clinically for minor surgical procedure

B-lymphocytes from Malignant Hyperthermia-susceptible Patients Have an Increased Sensitivity to Skeletal Muscle Ryanodine Receptor Activators

Malignant hyperthermia (MH) is a pharmacogenetic disease triggered by volatile anesthetics and succinylcholine in genetically predisposed individuals. The underlying feature of MH is a hypersensitivity of the calcium release machinery of the sarcoplasmic reticulum, and in many cases this is a result of point mutations in the skeletal muscle ryanodine receptor calcium release channel (RYR1). RYR1 is mainly expressed in skeletal muscle, but a recent report demonstrated the existence of this isoform in human B-lymphocytes. As B-cells can produce a number of cytokines, including endogenous pyrogens, we investigated whether some of the symptoms seen during MH could be related to the involvement of the immune system. Our results show that (i) Epstein-Barr virus-immortalized B-cells from MH-susceptible individuals carrying the V2168M RYR1 gene mutation were more sensitive to the RYR activator 4-chloro-m-cresol and (ii) their peripheral blood leukocytes produce more interleukin (IL)-1beta after treatment with the RYR activators caffeine and 4-chloro-m-cresol, compared with cells from healthy controls. Our result demonstrate that RYR1-mediated calcium signaling is involved in release of IL-1beta from B-lymphocytes and suggest that some of the symptoms seen during an MH episode may be due to IL-1beta production

Active Antigen-specific Immunotherapy of Melanoma: from Basic Science to Clinical Investigation

Advanced-stage melanoma here dismal prognosis, and novel therapeutic approaches are urgently required. The possibility of taking advantage of the immune response of patients for its treatment has been an appealing concept for almost a century. Only during the last decade, however, has the molecular identification of tumor-associated antigens (TAAs) offered the possibility of vaccinating patients (e.g., active induction of TAA-specific immune responses). Active antigen-specific immunotherapy (AASIT) is currently being investigated in a number of clinical centers as a treatment option for advanced-stage melanoma. A large number of melanoma TAAs have been molecularly characterized and are being used in vaccination trials in various molecular forms and according to various immunization protocols. Here we provide a short overview on melanoma TAAs, the technologies currently in use to induce specific cytotoxic T-lymphocyte (CTL) responses in vivo, and their monitoring. We also propose a tentative AASIT agenda for the next few years, aiming at improving the capacity to induce and monitor TAA-specific immune responses and to verify their clinical effectivenes

Effects of Elevated beta-Estradiol Levels on the Functional Morphology of the Testis - New Insights

Elevated estradiol levels are correlated with male infertility. Causes of hyperestrogenism include diseases of the adrenal cortex, testis or medications affecting the hypothalamus-pituitary-gonadal axis. The aim of our study was to elucidate the effects of estradiol treatment on testicular cellular morphology and function, with reference to the treatment regimen received. Testes samples (n = 9) were obtained post-orchiectomy from male-to-female transsexuals within the age range of 26–52 years. Each patient had a minimum of 1–6 years estradiol treatment. For comparison, additional samples were obtained from microscopically unaltered testicular tissue surrounding tumors (n = 7). The tissues obtained were investigated by stereomicroscopy, histochemistry, scanning electron microscopy (SEM) and immunohistochemistry. Our studies revealed that estradiol treatment significantly decreased the diameter of the seminiferous tubules (p < 0.05) and induced fatty degeneration in the surrounding connective tissue. An increase in collagen fiber synthesis in the extracellular matrix (ECM) surrounding the seminiferous tubules was also induced. Spermatogenesis was impaired resulting in mainly spermatogonia being present. Sertoli cells revealed diminished expression of estrogen receptor alpha (ERα). Both Sertoli and Leydig cells showed morphological alterations and glycoprotein accumulations. These results demonstrate that increased estradiol levels drastically impact the human testis

Whole blood assessment of antigen specific cellular immune response by real time quantitative PCR: a versatile monitoring and discovery tool

BACKGROUND: Monitoring of cellular immune responses is indispensable in a number of clinical research areas, including microbiology, virology, oncology and autoimmunity. Purification and culture of peripheral blood mononuclear cells and rapid access to specialized equipment are usually required. We developed a whole blood (WB) technique monitoring antigen specific cellular immune response in vaccinated or naturally sensitized individuals. METHODS: WB (300 microl) was incubated at 37 degrees C with specific antigens, in the form of peptides or commercial vaccines for 5-16 hours. Following RNAlater addition to stabilize RNA, the mixture could be stored over one week at room temperature or at 4 degrees C. Total RNA was then extracted, reverse transcribed and amplified in quantitative real-time PCR (qRT-PCR) assays with primers and probes specific for cytokine and/or chemokine genes. RESULTS: Spiking experiments demonstrated that this technique could detect antigen specific cytokine gene expression from 50 cytotoxic T lymphocytes (CTL) diluted in 300 microl WB. Furthermore, the high sensitivity of this method could be confirmed ex-vivo by the successful detection of CD8+ T cell responses against HCMV, EBV and influenza virus derived HLA-A0201 restricted epitopes, which was significantly correlated with specific multimer staining. Importantly, a highly significant (p = 0.000009) correlation between hepatitis B surface antigen (HBsAg) stimulated IL-2 gene expression, as detectable in WB, and specific antibody titers was observed in donors vaccinated against hepatitis B virus (HBV) between six months and twenty years before the tests. To identify additional markers of potential clinical relevance, expression of chemokine genes was also evaluated. Indeed, HBsAg stimulated expression of MIP-1beta (CCL4) gene was highly significantly (p = 0.0006) correlated with specific antibody titers. Moreover, a longitudinal study on response to influenza vaccine demonstrated a significant increase of antigen specific IFN-gamma gene expression two weeks after immunization, declining thereafter, whereas increased IL-2 gene expression was still detectable four months after vaccination. CONCLUSION: This method, easily amenable to automation, might qualify as technology of choice for high throughput screening of immune responses to large panels of antigens from cohorts of donors. Although analysis of cytokine gene expression requires adequate laboratory infrastructure, initial antigen stimulation and storage of test probes can be performed with minimal equipment and time requirements. This might prove important in "field" studies with difficult access to laboratory facilities

In Vitro Modeling of Tumor-Immune System Interaction.

Immunotherapy has emerged during the past two decades as an innovative and successful form of cancer treatment. However, frequently, mechanisms of actions are still unclear, predictive markers are insufficiently characterized, and preclinical assays for innovative treatments are poorly reliable. In this context, the analysis of tumor/immune system interaction plays key roles, but may be unreliably mirrored by in vivo experimental models and standard bidimensional culture systems. Tridimensional cultures of tumor cells have been developed to bridge the gap between in vitro and in vivo systems. Interestingly, defined aspects of the interaction of cells from adaptive and innate immune systems and tumor cells may also be mirrored by 3D cultures. Here we review in vitro models of cancer/immune cell interaction and we propose that updated technologies might help develop innovative treatments, identify biologicals of potential clinical relevance, and select patients eligible for immunotherapy treatments

Outcome Prediction for SARS-CoV-2 Patients Using Machine Learning Modeling of Clinical, Radiological, and Radiomic Features Derived from Chest CT Images

Featured Application The present study demonstrates that semi-automatic segmentation enables the identification of regions of interest affected by SARS-CoV-2 infection for the extraction of prognostic features from chest CT scans without suffering from the inter-operator variability typical of segmentation, hence offering a valuable and informative second opinion. Machine Learning methods allow identification of the prognostic features potentially reusable for the early detection and management of other similar diseases. (1) Background: Chest Computed Tomography (CT) has been proposed as a non-invasive method for confirming the diagnosis of SARS-CoV-2 patients using radiomic features (RFs) and baseline clinical data. The performance of Machine Learning (ML) methods using RFs derived from semi-automatically segmented lungs in chest CT images was investigated regarding the ability to predict the mortality of SARS-CoV-2 patients. (2) Methods: A total of 179 RFs extracted from 436 chest CT images of SARS-CoV-2 patients, and 8 clinical and 6 radiological variables, were used to train and evaluate three ML methods (Least Absolute Shrinkage and Selection Operator [LASSO] regularized regression, Random Forest Classifier [RFC], and the Fully connected Neural Network [FcNN]) for their ability to predict mortality using the Area Under the Curve (AUC) of Receiver Operator characteristic (ROC) Curves. These three groups of variables were used separately and together as input for constructing and comparing the final performance of ML models. (3) Results: All the ML models using only RFs achieved an informative level regarding predictive ability, outperforming radiological assessment, without however reaching the performance obtained with ML based on clinical variables. The LASSO regularized regression and the FcNN performed equally, both being superior to the RFC. (4) Conclusions: Radiomic features based on semi-automatically segmented CT images and ML approaches can aid in identifying patients with a high risk of mortality, allowing a fast, objective, and generalizable method for improving prognostic assessment by providing a second expert opinion that outperforms human evaluation

Expression of MAGE-A1, MAGE-A3/4 and NY-ESO-1 Cancer-Testis Antigens in Fetal Testis

SUMMARY Immunohistochemical expression of MAGE-A1, MAGE-A3/4 and NY-ESO-1/LAGE-1 cancer testis antigens (CTA) was assessed in 24 fetal testes from 15 th to 36 th week of gestation. Three monoclonal antibodies were used for immunohistochemical staining: 77B recognizing MAGE-A1, 57B recognizing multiple MAGE-A CTA, and D8.38 recognizing NY-ESO-1/ LAGE-1. Expression of MAGE-A1 was not observed in fetal testis samples, whereas multi-MAGE-A and NY-ESO-1/LAGE-1 specific reagents stained pro-spermatogonia in all samples with different expression levels during the period of fetal development observed. Significant expression of MAGE-A3/4 and almost continuous expression of NY-ESO-1 in fetal testes after 22 nd week of gestation suggested their important role in the development of sex cords and pro-spermatogonia in particular